Pancreatic ductal proliferation is a frequent hallmark of surgical pancreatic disease. However, the epithelial stem cells responsible for pancreatic duct proliferation remain unknown. The long-term objective of this research program is to identify the "cell of origin" from which ductal proliferation originates during chronic pancreatitis and pancreatic cancer, potentially allowing therapeutic intervention to be directed to precursor cells. Using both surgical and transgenic models of ductal proliferation, preliminary experiments have been performed suggesting that pancreatic stem cells can be identified based on the following criteria: 1) ductal location, 2) enhanced proliferative capacity, 3) expression of the anti-apoptotic protein Bcl-2, and 4) expression of the Pdx1 and Pax6 homeobox transcription factors. Based on these studies, the following specific aims are proposed: First, to characterize the role of Pdx1- and Pax6- expressing pancreatic stem cells in benign pancreatic ductal proliferation induced by surgical pancreatic duct ligation. Second, to characterize the role of Pdx1- and Pax6-expressing stem cells in premalignant pancreatic duct proliferation induced by TGFalpha overexpression in MT-TGFalpha transgenic mice. Third, to isolate and propagate Pdx1-expressing pancreatic stem cells in long-term tissue culture, and to investigate the effects of TGFalpha and transforming oncogenes on their growth and differentiation. These investigations will be pursued utilizing a novel in vivo reporter gene approach which takes advantage of pre-existing mouse lines in which one copy of either the endogenous Pdx1 locus or the endogenous Pax6 locus has been replaced with a lacZ cassette. This allows precise tracing of candidate stem cells expressing these homeobox genes. In addition, a strategy for isolation of pancreatic stem cells is proposed using the Pdx1 promoter to target expression of the neomycin antibiotic resistance gene to the embryonic pancreatic ductal epithelium. Together, these investigations will provide important new insight regarding the pancreatic cell lineages participating in ductal proliferation, and potentially lead to novel therapeutic strategies for surgical pancreatic disease.